Identifying the rate-limiting step in the catalytic mechanism of indole-3-glycerol phosphate synthase in Mycobacterium Tuberculosis
Presentation Type
Abstract
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
25-4-2025 9:00 AM
End Date
25-4-2025 9:59 AM
Description
M. tuberculosis (Mt) is the bacterial cause of a leading infectious disease, tuberculosis (TB). The efficacy of first line antitubercular drugs has diminished due to the increasing presence of Mt drug resistance The enzyme indole-3-glycerol phosphate synthase (IGPS) in Mt (MtIGPS) was proposed to be a potential drug target due to its role in catalyzing the fourth step of tryptophan biosynthesis, a pathway essential for the survival of Mt. MtIGPS catalyzes the ring closure in 1-(o-carboxylphenylamino)-1-deoxyribulose 5-phosphate (CdRP) to form indole-3-glycerol phosphate (IGP). Understanding how MtIGPS operates and determining the rate-limiting step of the MtIGPS mechanism is essential for predicting potential inhibitors that specifically target the MtIGPS-CdRP interaction. Wildtype and E168D MtIGPS enzymes were expressed, purified, and used in kinetic assays and stopped flow experiments to determine kinetic parameters and rate-limiting steps. Change in fluorescence intensity over time was compared in multiple versus single turnover conditions to obtain kcat and k2 values, and rate ratios were obtained to compare activity in H2O and D2O-based buffers for wildtype MtIGPS. E168D revealed a 480-fold reduction in catalytic activity compared to wildtype. This result, in addition to an alteration in pH profiles, suggests that E168 is a catalytically important residue. It was hypothesized that E168 plays a role in stabilizing the positively charged nitrogen of the first intermediate in the mechanism or it may serve as a catalytic base in the dehydration step. Wildtype MtIGPS showed similar kcat and k2 values, indicating that the rate-limiting step lies within the chemical conversion of CdRP to IGP, characterized by the k2 value. Activity in the presence of D2O significantly reduced at pH 7.5 and results of rate comparison in H2O versus D2O will be further explored at various pH to determine if the rate-limiting step is a proton transfer step and to determine the effect of pH. Together, these results contribute to an enhanced understanding of MtIGPS catalysis which is useful in the design of mechanism-based inhibitors.
Identifying the rate-limiting step in the catalytic mechanism of indole-3-glycerol phosphate synthase in Mycobacterium Tuberculosis
M. tuberculosis (Mt) is the bacterial cause of a leading infectious disease, tuberculosis (TB). The efficacy of first line antitubercular drugs has diminished due to the increasing presence of Mt drug resistance The enzyme indole-3-glycerol phosphate synthase (IGPS) in Mt (MtIGPS) was proposed to be a potential drug target due to its role in catalyzing the fourth step of tryptophan biosynthesis, a pathway essential for the survival of Mt. MtIGPS catalyzes the ring closure in 1-(o-carboxylphenylamino)-1-deoxyribulose 5-phosphate (CdRP) to form indole-3-glycerol phosphate (IGP). Understanding how MtIGPS operates and determining the rate-limiting step of the MtIGPS mechanism is essential for predicting potential inhibitors that specifically target the MtIGPS-CdRP interaction. Wildtype and E168D MtIGPS enzymes were expressed, purified, and used in kinetic assays and stopped flow experiments to determine kinetic parameters and rate-limiting steps. Change in fluorescence intensity over time was compared in multiple versus single turnover conditions to obtain kcat and k2 values, and rate ratios were obtained to compare activity in H2O and D2O-based buffers for wildtype MtIGPS. E168D revealed a 480-fold reduction in catalytic activity compared to wildtype. This result, in addition to an alteration in pH profiles, suggests that E168 is a catalytically important residue. It was hypothesized that E168 plays a role in stabilizing the positively charged nitrogen of the first intermediate in the mechanism or it may serve as a catalytic base in the dehydration step. Wildtype MtIGPS showed similar kcat and k2 values, indicating that the rate-limiting step lies within the chemical conversion of CdRP to IGP, characterized by the k2 value. Activity in the presence of D2O significantly reduced at pH 7.5 and results of rate comparison in H2O versus D2O will be further explored at various pH to determine if the rate-limiting step is a proton transfer step and to determine the effect of pH. Together, these results contribute to an enhanced understanding of MtIGPS catalysis which is useful in the design of mechanism-based inhibitors.
Comments
Poster presentation at the 2025 Student Research Symposium.